It is known that pathological neovascularization is closely related to the symptoms or causes of certain diseases. Solid tumors are representative of such diseases. For the growth of tumor tissue beyond the diameter of 1 to 2 mm, newly formed blood vessels need to extend from the existing blood vessels to reach the tumor tissue (J. Folkman, J. Natl. Cancer Inst., 82:4 (1990)). When the blood vessel reaches the tumor tissue, its growth is explosively accelerated. Diabetic retinopathy is accompanied by pathological neovascularization of the retina, which often leads to the loss of eyesight. Moreover, pathological neovascularization is also seen in such diseases as chronic rheumatoid arthritis, psoriasis, hemangioma, scleroderma, and neovascular glaucomas, and it is considered to be one of the main symptoms (J. Folkman and N. Engle, J. Med., 320:1211 (1989)). Therefore, it may be possible to use substances that inhibit neovascularization for the treatment of tumors and other diseases mentioned above.
Vascular endothelial cells are the cells that constitute the innermost layer of the blood vessel. Neovascularization occurs when vascular endothelial cells proliferate upon stimulation by growth factors, physiologically active substances, or mechanical damages. Known growth factors that can directly or indirectly stimulate the proliferation of vascular endothelial cells include bFGF (basic Fibroblast Growth Factor), aFGF (acidic Fibroblast Growth Factor), VEGF (Vascular Endothelial cell Growth Factor), PD-ECGF (Platelet-Derived Endothelial Cell Growth Factor), TNF-.alpha. (Tumor Necrosis Factor-.alpha.), PDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), TGF-.alpha. (Transforming Growth Factor-.alpha.), and HGF (Hepatocyte Growth Factor) (L. Diaz-Flores et al., Histol. Histopath., 9:807 (1994)). Particularly, VEGF can be distinguished from the other growth factors by the fact that its action is very specific to vascular endothelial cells. In other words, the VEGF receptor is found in very few cells other than vascular endothelial cells.
VEGF is a glycoprotein whose molecular weight is 40,000-45,000, and exists as a dimer (P. W. Leung et al., Science, 246:1306 (1989), P. J. Keck et al., Science, 246: 1319 (1989)). VEGF acts, by binding to the VEGF receptor, to promote cell proliferation and enhance membrane permeability.
The following reports suggest the involvement of VEGF in tumor. Many tumor cells secrete VEGF (S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). When tumor tissue sections are stained with an anti-VEGF antibody, the tumor tissue is strongly stained as well as the newly formed blood vessels surrounding it (H. F. Dvorak et al., J. Exp. Med. 174:1275 (1991), L. F. Brown et al., Cancer Res., 53:4727 (1993)). Growth of a transplanted tumor is suppressed in mice in which one of the VEGF receptors is genetically inactivated (B. Millauer et al., Nature, 367:576 (1994)). Anti-VEGF neutralizing antibodies exhibit anti-tumor activities in tumor-bearing mice (K. J. Kim et al., Nature, 362:841 (1993), S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). From these facts, it is considered that VEGF secreted by tumor cells plays a major role in neoplastic neovascularization.
In humans there are two known VEGF receptors, FLT (M. Shibuya et al., Oncogene, 5:519 (1990)) and KDR (B. I. Terman et al., Biochem. Biophys. Res. Commun., 187:1579 (1992)). The extracellular domain of FLT (also known in the art as FLT-1) has seven immunoglobulin-like domains as shown in FIG. 1 (C. DeVries et al., Science, 255:989 (1992)). Regarding FLT, a cDNA of a soluble-type receptor has been cloned (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci. U.S.A., 90:10705 (1993)). The polypeptide encoded by this cDNA corresponds to the first through sixth immunoglobulin-like domains of the seven immunoglobulin-like domains of the FLT extracellular domain, and it inhibited the VEGF activities by binding to VEGF with an affinity comparable to that of the full-length FLT. Regarding KDR, it is also known that the genetically engineered first through sixth immunoglobulin-like domains of the extracellular domain bind to VEGF (R. L. Kendal et al., Biochem. Biophys. Res. Commun., 201:326(1994)).